Critical Role of microRNA-33a/b in Cardiovascular and Metabolic Disease: Molecular Mechanisms and Therapeutic Perspectives

Abstract

MicroRNAs (miRNAs) serve as fundamental post-transcriptional regulators of gene expression, among which the miR-33 family, consisting of miR-33a and miR-33b, has emerged as a critical modulator in the pathogenesis of cardiovascular and metabolic diseases. These miRNAs are embedded within the intronic regions ofSREBF genes and play pivotal roles in cholesterol homeostasis, fatty acid metabolism, and inflammatory regulation. Notably, miR-33a is highly conserved across various species, whereas miR-33b is found primarily in primates and some other mammals, complicating the development of relevant animal models. These miRNAs inhibit their target genes involved in cholesterol metabolism, fatty acid oxidation, and insulin signaling, consequently influencing the development and progression of cardiovascular and metabolic diseases. Inhibition or genetic ablation of miR-33 has shown therapeutic potential, improving dyslipidemia, atherosclerosis, and metabolic dysfunction-associated steatotic liver disease, through altered cholesterol metabolism, attenuation of inflammation, and increased fatty acid utilization. In addition, miR-33 suppression has been shown to promote skeletal muscle regeneration. However, systemic inhibition of miR-33 requires caution due to the role of miR-33 in hunger signaling and sympathetic nerve activity in the central nervous system, which may lead to obesity. Therefore, the development of tissue-specific strategies is essential for the safe and effective therapeutic targeting of miR-33.

Introduction

Cardiovascular and metabolic diseases, such as atherosclerosis, dyslipidemia, type 2 diabetes mellitus (T2DM), obesity, and metabolic dysfunction-associated steatotic liver disease (MASLD), are among the leading causes of morbidity and mortality worldwide ^{1 , 2)} . These disorders are fueled by overlapping metabolic dysfunctions, including dyslipidemia, insulin resistance, hypertension, obesity, and inflammation. Despite significant advances in pharmacological therapies targeting glucose and lipid metabolism, the residual risk remains high, especially in patients with mixed metabolic profiles.

In recent years, non-coding RNAs, particularly microRNAs (miRNAs), have emerged as key regulators of gene expression in both physiological and pathological conditions. MiRNAs are small, endogenous, non-coding RNAs, approximately 21–25 nucleotides in length, that bind to the 3’-untranslated regions (3’-UTRs) of target messenger RNAs (mRNAs), leading to translational repression or mRNA degradation. Since their discovery in Caenorhabditis elegans in 1993 by the research groups of Victor Ambros and Gary Ruvkun ^{3 , 4)} , miRNAs have been recognized as crucial post-transcriptional regulators involved in development, metabolism, immunity, and cancer.

Among the thousands of miRNAs identified to date, estimated in over 2,500 mature species in humans, the miR-33 family has gained particular attention in the context of metabolic diseases ^{5 - 8)} . The miR-33 family, consisting of the miR-33a and miR-33b isoforms, plays a pivotal role in the regulation of lipid metabolism, cholesterol homeostasis, insulin signaling, fatty acid oxidation, and inflammatory pathways. The miR-33 family members are located within the intronic regions of genes that control lipid metabolism, including sterol regulatory element-binding factor (SREBF) 2 for miR-33a and SREBF1 for miR-33b. This genomic arrangement facilitates the coordinated transcriptional and post-transcriptional regulation of key metabolic target genes. This unique genomic architecture establishes a robust regulatory network, positioning the miR-33 family as a key orchestrator of cellular lipid homeostasis.

This review delves into the biology of miR-33a and miR-33b and highlights their roles in cardiovascular and metabolic diseases. We examine their molecular mechanisms, dissect their regulatory networks, and consider their potential for therapeutic use.

Genomic Organization and Conservation of miR-33a and miR-33b

miR-33a and miR-33b are embedded within the intronic regions of SREBF genes. These genes encode sterol regulatory element-binding proteins (SREBPs), which act as central transcriptional regulators of lipid biosynthesis ⁹⁾ . MiR-33a resides within intron 16 of SREBF2, which is primarily involved in the regulation of cholesterol homeostasis genes, such as low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). In contrast, miR-33b is located within intron 17 of SREBF1, which regulates the expression of genes related to fatty acid and triglyceride homeostasis, such as fatty acid synthase (FASN), acetyl-CoA carboxylase α (ACACA) and stearoyl-CoA desaturase 1 (SCD1) ^{6 - 8)} . Although miR-33a and miR-33b differ by only two nucleotides, they share the same seed sequences, and their functions have been found to be relevant through comprehensive target analysis ¹⁰⁾ . Notably, miR-33a is highly conserved across a broad range of species, including rodents, whereas miR-33b is only found primarily in primates and some other mammals, being entirely absent in commonly used rodent models, such as mice and rats. This evolutionary divergence complicates the modeling of the miR-33b function in these animals.

Nevertheless, the presence of both isoforms in primates indicates an additional layer of complexity in the metabolic regulation in humans. The expression of miR-33 is closely associated with that of its host genes. When SREBFs are trans-activated by metabolic stimuli, such as low intracellular cholesterol (for SREBF2), insulin, or high carbohydrate intake (for SREBF1), both the host gene and the embedded miR-33 are co-transcribed. This co-transcription enables miR-33 to fine-tune the SREBP pathway by repressing genes that are involved in lipid metabolism, thereby ensuring a balanced metabolic response. Their key features and representative target genes in cardiovascular and metabolic diseases are summarized in Table 1 .

Table 1. Key features and representative target genes of miR-33a and miR-33b in cardiovascular and metabolic diseases.

	miR-33a	miR-33b	References
Host gene	SREBF2	SREBF1
- Primary function	Cholesterol synthesis and uptake	Fatty acid synthesis
- Location of miRNA	Intron 16	Intron 17
Species Conservation	Highly conserved across species, including rodents	Primates and some other mammals; absent in rodents	5-8
Preclinical models	Wild-type mice, miR-33a deficient mice, non-human primates	miR-33b knock-in mice, non-human primates	11-14, 16-18, 40
Key target genes	Target genes are largely shared, because the seed sequences are same.		10
	- Cholesterol metabolism and atherosclerosis: ABCA1, NPC1, OSBPL6, PRKAA1		11-19, 21-33
	- Fatty acid oxidation: CPT1A, HADHB, CROT, PRKAA1		15, 17, 34-38
	- Insulin signaling and energy homeostasis: IRS2, SIRT6, PRKAA1, PPARGC1A		34, 35
	- Food intake and hunger signaling: PRKAA1, PPARGC1A, CPT1a, CROT		39, 40, 41
	- Energy expenditure and GABAA signaling: GABRB2, GABRA4		43
	- Skeletal muscle regeneration: CDK6, FST, ABCA1		45

Cholesterol Homeostasis and Dyslipidemia

The most well-known target gene of miR-33a/b is ATP-binding cassette subfamily A member 1 (ABCA1). ABCA1 is a cholesterol transporter that facilitates the ATP-dependent transfer of intracellular cholesterol to extracellular lipid-poor apolipoprotein A (ApoA)-1, leading to the formation of preβ-high-density lipoprotein (preβ-HDL). In miR-33a knockout mice or when miR-33a is suppressed by synthetic nucleic acids such as antagomirs, the expression of ABCA1 in macrophages and the liver is increased ^{11 - 15)} . Consequently, this enhances the cholesterol efflux capacity of macrophages and elevates serum HDL cholesterol levels. Evaluating the function of miR-33b in mice presents challenges as described above; however, miR-33b knock-in mice have been generated by inserting the human miR-33b sequence into the same Srebf1 intron as found in humans ¹⁶⁾ . In these mice, both miR-33a and miR-33b are expressed, reflecting the human condition. In contrast to miR-33a knockout mice, miR-33b knock-in mice exhibit a decreased ABCA1 expression in macrophages and the liver, leading to a reduced cholesterol efflux capacity in macrophages and decreased serum HDL cholesterol levels. These findings have also been validated in non-human primates, suggesting that miR-33a and miR-33b play crucial roles in cholesterol regulation in humans ^{17 , 18)} . Other studies have identified Niemann-Pick disease, type C1 (NPC1), and oxysterol-binding protein-like 6 (OSBPL6) as additional target genes of miR-33, which can affect the cholesterol metabolism and efflux capacity ^{12 , 19)} .

Atherosclerosis

Atherosclerosis is a chronic inflammatory condition of the arterial wall characterized by cholesterol deposition, immune cell infiltration, and plaque formation; therefore, miR-33a/b significantly influence the development of atherosclerosis. HDL cholesterol is known to have anti-inflammatory and anti-atherosclerotic properties. In addition, it also exerts an anti-atherosclerotic effect by promoting reverse cholesterol transport (RCT), which returns excessive peripheral cholesterol to the liver ²⁰⁾ . Inhibition or genetic ablation of miR-33a has been shown to either promote the regression of atherosclerosis or prevent its formation in mouse models, such as LDL receptor-deficient or apoprotein E (ApoE)-deficient mice, conferring resistance to the disease ^{21 - 23)} . Furthermore, macrophages within the atherosclerotic lesions of these mice exhibit an anti-inflammatory phenotype characterized by enhanced cholesterol efflux, which leads to a reduction in foam cell formation. MiR-33a knockout in mice has also been reported to suppress the formation of abdominal aortic aneurysm (AAA) by increasing serum HDL cholesterol and promoting an anti-inflammatory response in both macrophages and vascular smooth muscle cells ²⁴⁾ . Given the critical role of macrophages in atherosclerotic diseases, their relationship with miR-33a has been extensively studied. Inhibition of miR-33a or miR-33a deletion in macrophages has been shown to affect myeloid cell differentiation ²⁵⁾ , induce M2 macrophage polarization with mitochondrial respiratory and metabolic changes ^{26 , 27)} , and enhance autophagy ^{28 , 29)} . Single-cell analyses have confirmed their anti-inflammatory and profibrotic effects in macrophages, which can contribute to atheroprotective effects and plaque stabilization ^{30 , 31)} .

Conversely, studies on miR-33b have been conducted using miR-33b knock-in mice. In ApoE-deficient mice, miR-33b knock-in promotes an inflammatory shift in macrophages by promoting foam cell formation, impairing reverse cholesterol transport through reduced HDL cholesterol levels, accelerating the progression of atherosclerosis, and notably increasing the formation of unstable plaques ³²⁾ . It also exacerbates the formation of AAA, whereas the inhibition of miR-33b with synthetic nucleic acids suppresses their progression ³³⁾ . These results suggest that miR-33a/b inhibition may be a valuable therapeutic strategy for atherosclerotic diseases.

Fatty Acid Oxidation

In addition to its effects on cholesterol metabolism, miR-33 also regulates fatty acid oxidation. MiR-33 suppresses genes involved in mitochondrial β-oxidation, such as carnitine palmitoyltransferase 1A (CPT1A), hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit b (HADHB), and carnitine O-octanoyltransferase (CROT) ^{15 , 17 , 34 , 35)} . These enzymes are critical for the transport and breakdown of long-chain fatty acids in the mitochondria. By repressing these genes, miR-33 reduces the capacity for fatty acid oxidation, thereby promoting lipid accumulation in the liver. Conversely, miR-33 inhibition is considered to enhance fatty acid utilization and have an impact on steatosis treatment, as discussed in the next section.

Metabolic Dysfunction-associated Steatoic Liver Disease (MASLD)

MASLD includes a spectrum of hepatic abnormalities, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH). Dysregulation of lipid metabolism and insulin resistance is central to its pathogenesis. MiR-33 contributes to hepatic lipid accumulation by repressing fatty acid oxidation genes and promoting lipogenesis. Several studies have been conducted using steatohepatitis models. MiR-33a knockout in hepatocytes reduces steatosis with improved insulin sensitivity and glucose homeostasis and suppresses the subsequent progression to MASH and hepatocellular carcinoma (HCC) by reducing the lipid burden in the liver ^{36 , 37)} . Hepatic deletion of miR-33a reduces liver lipid content by suppressing lipid synthesis and enhancing fatty acid oxidation, improves the mitochondrial function, and alleviates oxidative stress through AMPKα and PGC1α activation, leading to reduced activation of the YAP/TAZ pathway. Furthermore, studies using miR-33b knock-in mice have reported that miR-33b promotes steatohepatitis through its effects on hepatocytes or hepatic stellate cells and that its inhibition by synthetic nucleic acids suppresses steatohepatitis formation by reducing cholesterol and fatty acid accumulation ³⁸⁾ . These data highlight the critical roles of miR-33a and miR-33b in the progression from MASLD to MASH and ultimately to HCC.

Insulin Signaling and Glucose Metabolism

miR-33 also influences insulin signaling and glucose homeostasis. It represses insulin receptor substrate 2 (IRS2), protein kinase AMP-activated catalytic subunit a1 (PRKAA1), and sirtuin 6 (SIRT6) ^{34 , 35)} , genes that are important for the maintenance of insulin sensitivity, cellular energy balance, and the mitochondrial function.

Obesity and Metabolic Syndrome

Obesity is a multifactorial disorder characterized by excessive lipid accumulation in the adipose tissue, systemic inflammation, and metabolic dysregulation. MiR-33a knockout mice exhibit obesity and insulin resistance when fed a high-fat diet ^{39 , 40)} . This has been attributed to a central nervous system phenotype, specifically an increase in food intake. MiR-33a has been shown to control hunger signaling in hypothalamic AgRP neurons possibly through several miR-33 target genes such as PRKAA1, Ppargc1a, CPT1a and CROT; thus, loss of miR-33a leads to increased feeding, obesity, and metabolic dysfunction in mice ⁴¹⁾ . In addition, miR-33a has been shown to suppress several gamma-aminobutyric acid (GABA)-related proteins, thereby regulating state-dependent memory ⁴²⁾ . MiR-33a deficiency decreases sympathetic nervous activity through the activation of GABAergic inhibitory neurons, mediated by the enhanced expression of GABA_A receptor subunits, such as GABRB2 and GABRA4, which leads to reduced adaptive thermogenesis primarily in brown adipose tissue (BAT) and contributes to obesity ⁴³⁾ . In contrast, studies focusing solely on BAT have reported that inhibition of miR-33a enhances its activity by upregulating UCP1-associated target genes, such as Znf516, Dio2, and Ppargc1a ⁴⁴⁾ . This suggests that the balance between the central nervous system and the peripheral tissues is of critical importance.

Skeletal Muscle Regeneration and Muscular Dystrophy

Recently, miR-33a/b was found to be abundantly expressed in skeletal muscle tissues and to be important for muscular regeneration. In muscle stem cells, known as satellite cells, miR-33a/b suppress the expression of genes crucial for muscle regeneration and maturation, such as cyclin-dependent kinase 6 (CDK6), follistatin (FST), and ABCA1 ^{45 , 46)} . MiR-33a knockout mice show enhanced skeletal muscle regeneration, and when crossbred with Duchene muscular dystrophy model mice (mdx mice), they exhibit ameliorated muscle damage and increased exercise tolerance. In contrast, miR-33b knock-in mice show exacerbated muscle damage and reduced exercise tolerance. The administration of synthetic nucleic acids to inhibit miR-33a/b promotes muscle regeneration, suppresses tissue fibrosis, and improves the muscle function. The skeletal muscle is a key organ in systemic metabolism and can therefore influence whole-body metabolic homeostasis.

Conclusion and Future Perspectives

miR-33a and miR-33b have been recognized as key regulators of lipid metabolism, glucose homeostasis, the mitochondrial function, and inflammation. Their coordinated interaction with SREBP transcription factors facilitates precise regulation of energy homeostasis and cellular lipid status. However, the dysregulated expression of miR-33a and miR-33b is implicated in the pathogenesis of cardiovascular and metabolic diseases, including dyslipidemia, atherosclerosis, MASLD, insulin resistance, obesity, and macrophage dysfunction. Inhibition of miR-33 in preclinical models considerably improves metabolic outcomes and ameliorates disease phenotypes, providing a strong rationale for its therapeutic development. Nonetheless, several challenges remain to be addressed, including tissue specificity, isoform selectivity, long-term safety, and interspecies differences, particularly the fact that miR-33b is present primarily in primates and several other mammals but absent in commonly used rodent models, such as mice and rats. Therefore, non-human primate studies provide a critical translational bridge, offering insights into whole-organism pharmacodynamics, pharmacokinetics, and safety profiles that cannot be captured in rodents. However, the inherent limitations of such studies, including small sample sizes, high costs, and ethical constraints, underscore the need to integrate non-human primate data with complementary approaches, such as studies using in vitro human cells including iPS cells, humanized rodent models, and ex vivo human tissues, rather than relying solely on primate findings to predict clinical outcomes.

In addition, systemic inhibition poses problems, as deletion of miR-33a in the central nervous system paradoxically leads to obesity by altering hunger signaling and sympathetic nerve tone. Thus, the therapeutic potential of targeting miR-33 depends on the development of advanced tissue-specific strategies that deliver therapeutic effects to peripheral tissues while sparing the central nervous system. Recent advances, particularly GalNAc-mediated hepatocyte targeting and optimized ASO chemistries, have enabled safer and more tissue-restricted modulation in clinical settings. However, achieving effective delivery beyond the liver remains technically challenging, highlighting the importance of addressing technical limitations and ensuring safety. Taking these considerations into account, the selection of the most appropriate disease for initial clinical application of miR-33 inhibition, which is implicated in a wide range of diseases, will likely depend on the progress of future research.

In conclusion, targeting miR-33 represents a promising yet intricate strategy for the treatment of cardiovascular and metabolic diseases. Future research will determine whether this approach can be safely and effectively translated into clinical practice.

Acknowledgements

At the 57th Annual Meeting of the Japan Atherosclerosis Society, T.H. was honored with the Goto Yuichiro Award. This review summarizes the lecture content presented at that time. This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), the Japan Society for the Promotion of Science (JSPS) KAKENHI grants 17K09860, 20K08904, and 23K07988, the Japan Science and Technology Agency for the Fusion Oriented Research for Disruptive Science and Technology program (JST FOREST) grant JPMJFR235F, and the Japan Agency for Medical Research and Development (AMED) grant 24ek0210202h0001.

Competing Interests

None.